bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Murine deficiency of peroxisomal L-bifunctional protein (EHHADH) causes medium-chain 3-

hydroxydicarboxylic aciduria and perturbs hepatic cholesterol homeostasis

Pablo Ranea-Robles1, Sara Violante1,2, Carmen Argmann1, Tetyana Dodatko1, Dipankar Bhattacharya3,

Hongjie Chen1,4, Chunli Yu1,4, Scott L. Friedman3, Michelle Puchowicz5,6, Sander M. Houten1

1Department of Genetics and Genomic Sciences, Icahn Institute for Data Science and Genomic

Technology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

2The Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer

Center, New York, NY 10065, USA.

3Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New

York, NY 10029, USA.

4Mount Sinai Genomics, Inc, Stamford, CT 06902, USA.

5Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, OH 44106

USA.

6Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

Short title: The role of peroxisomal L-bifunctional protein

Address for correspondence: Sander Houten, Department of Genetics and Genomic Sciences, Icahn

School of Medicine at Mount Sinai, 1425 Madison Avenue, Box 1498, New York, NY 10029, USA.

Phone: +1 212 659 9222, E-mail: [email protected]

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Abstract

Peroxisomes play an essential role in the β-oxidation of dicarboxylic acids (DCAs), which are metabolites

formed upon ω-oxidation of fatty acids. Genetic evidence linking transporters and enzymes to specific

DCA β-oxidation steps is generally lacking. Moreover, the physiological functions of DCA metabolism

remain largely unknown. In this study, we aimed to characterize the DCA β-oxidation pathway in human

cells, and to evaluate the biological role of DCA metabolism using mice deficient in the peroxisomal L-

bifunctional protein (Ehhadh KO mice). In vitro experiments using HEK-293 KO cell lines demonstrate

that ABCD3 and ACOX1 are essential in DCA β-oxidation, whereas both the bifunctional proteins

(EHHADH and HSD17B4) and the thiolases (ACAA1 and SCPx) have overlapping functions and their

contribution may depend on expression level. We also show that medium-chain 3-hydroxydicarboxylic

aciduria is a prominent feature of EHHADH deficiency in mice most notably upon inhibition of

mitochondrial fatty acid oxidation. Using stable isotope tracing methodology, we confirmed that products

of peroxisomal DCA β-oxidation can be transported to mitochondria for further metabolism. Finally, we

show that, in liver, Ehhadh KO mice have increased mRNA and protein expression of cholesterol

biosynthesis enzymes with decreased (in females) or similar (in males) rate of cholesterol synthesis. We

conclude that EHHADH plays an essential role in the metabolism of medium-chain DCAs and postulate

that peroxisomal DCA β-oxidation is a regulator of hepatic cholesterol biosynthesis.

Keywords multifunctional enzyme 1; fatty acid oxidation; omega-oxidation; dodecanedioic acid; hexadecanedioic acid

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Introduction

Fatty acid β-oxidation (FAO) constitutes the primary source of energy during metabolically

demanding conditions such as fasting or exercise (Houten et al. 2016). The major FAO pathway is

mitochondrial, but peroxisomes also harbor FAO pathways that metabolize specific carboxylic acids such

as very long-chain fatty acids, branched-chain fatty acids, bile acids and long-chain dicarboxylic acids

(DCAs) (Wanders & Waterham 2006). These DCAs are products of fatty acid ω-oxidation, an alternative

pathway for fatty acid metabolism initiated by microsomal cytochrome P450 enzymes of the CYP4A

family (Hardwick et al. 1987; PREISS & BLOCH 1964). CYP4A enzymes catalyze the ω-hydroxylation

of long-chain fatty acids that are subsequently converted into long-chain DCAs by the successive action

of an alcohol and an aldehyde dehydrogenase. Peroxisomal β-oxidation of DCAs generates unique

products such as adipic and succinic acid (Jin et al. 2015; Rusoff et al. 1960; Tserng & Jin 1991a), the

latter being able to enter the TCA cycle. In addition, peroxisomal β-oxidation yields acetyl-CoA units,

which have a variety of fates including lipogenesis, ketogenesis, or oxidation in the TCA cycle. These

metabolic pathway connections suggest an important role for DCA β-oxidation in intermediary

metabolism.

Mitochondrial and peroxisomal FAO as well as ω-oxidation are usually induced in parallel,

particularly in the liver and the kidney, under conditions of high lipid flux such as during fasting

(Mortensen & Gregersen 1981; Pettersen et al. 1972). Notably, medium-chain dicarboxylic aciduria; i.e.

the excretion of C6-, C8-, and C10-DCAs (adipic, suberic and sebacic acids, respectively) in the urine, is

a feature shared between fasting individuals (ketosis) and patients with inherited defects in mitochondrial

FAO (Fontaine et al. 1998; Gregersen et al. 1980; Karpati et al. 1975; Przyrembel et al. 1976; Röschinger

et al. 2000; Vianey-Saban et al. 1998). These findings further illustrate the important role of fatty acid ω-

oxidation and subsequent DCA β-oxidation under lipid metabolic stress. However, the physiological roles

of DCA metabolism remain largely to be established.

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DCA metabolism begins with their activation by a dicarboxylyl-CoA synthetase (Vamecq et al.

1985). Subsequent DCA β-oxidation is thought to occur in peroxisomes (Dirkx et al. 2007; Ferdinandusse

et al. 2004; Kølvraa & Gregersen 1986; Suzuki et al. 1989). Both EHHADH (also known as peroxisomal

L-bifunctional protein, LBP) and HSD17B4 (also known as D-bifunctional protein, DBP) can catalyze the

second and third steps of peroxisomal FAO. We and others have demonstrated that EHHADH plays a

prominent role in the oxidation of long-chain DCAs (Ding et al. 2013; Dirkx et al. 2007; Ferdinandusse et

al. 2004; Houten et al. 2012; Nguyen et al. 2008). Other peroxisomal proteins that participate in DCA

metabolism are the ABCD3 transporter (van Roermund et al. 2014), ACOX1 (acyl-CoA oxidase 1)

(Ferdinandusse et al. 2004; Poosch & Yamazaki 1989), and the thiolases ACAA1 (3-ketoacyl-CoA

thiolase) and SCPx (sterol carrier protein X) (Ferdinandusse et al. 2004). In many cases, genetic evidence

that links the encoding genes to DCA β-oxidation is lacking. In summary, ω-oxidation and subsequent

peroxisomal DCA β-oxidation is an alternate pathway for fatty acid metabolism that does not rely on the

classic mitochondrial FAO pathway.

In this study, we assessed the contribution of candidate peroxisomal proteins to DCA β-oxidation

using KO HEK-293 cell lines created by CRISPR-Cas9 genome editing. We also examined the

physiological role of DCA β-oxidation through the study of the Ehhadh KO mouse model. Finally, we

assessed the relevance of peroxisomal DCA metabolism in mouse models with mitochondrial FAO

defects. The results of our study further highlight an important role for peroxisomal DCA β-oxidation in

intermediary metabolism.

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Material and Methods

Materials

HEK-293 cells (ATCC® CRL-1573™) were obtained from the American Type Culture Collection

(Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Krebs–Henseleit buffer,

William’s E Medium with GlutaMAX™ Supplement (WEGG), heat-inactivated fetal bovine serum

(FBS), penicillin, streptomycin, and Lipofectamine 2000 were purchased from Thermo Fisher Scientific

(Waltham, MA, USA). CRISPR-Cas9 plasmid [pSpCas9(BB)-2A–green fluorescent protein (GFP);

PX458] (Ran et al. 2013) was obtained from Addgene (Watertown, MA, USA). The blocking buffer and

the fluorescent secondary anti-rabbit and anti-mouse IgG antibodies were obtained from LI-COR

2 2 Biosciences (Lincoln, NE, USA). [ H]-water (99% enriched), the L-carnitine internal standard ( H9-

2 2 carnitine) and the acylcarnitine internal standard mix containing H9-carnitine, H3-acetylcarnitine (C2),

2 2 2 2 H3-propionylcarnitine (C3), H3-butyrylcarnitine (C4), H9-isovalerylcarnitine (C5), H3-

2 2 octanoylcarnitine (C8), H9-myristoylcarnitine (C14), and H3-palmitoylcarnitine (C16), were from

Cambridge Isotope Laboratories (Tewksbury, MA, USA). Minimal essential medium, bovine serum

albumin (fatty acid free), L-carnitine, lauric acid (C12:0), [U-13C]-dodecanedioic acid ([U-13C]-C12-

DCA, 99 atom % 13C), dodecanedioic acid (C12-DCA), and hexadecanedioic acid (C16-DCA) were

obtained from MilliporeSigma (Burlington, MA, USA). L-Aminocarnitine [L-AC; (R)-aminocarnitine,

minimum 97%], 3-OH-adipic (3-hydroxyhexanedioic acid, 97%), 3-OH-suberic (3-hydroxyoctanedioic

acid, 97%), and 3-hydroxydodecanedioic (97%) were obtained from Toronto Research Chemical Inc.

(Toronto, ON, Canada). 3-OH-sebacic (>98%) was obtained from Santa Cruz (Dallas, TX). All other

chemicals were of analytical grade.

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Animals

All mouse experiments were approved by the Institutional Animal Care and Use Committee

(IACUC) of the Icahn School of Medicine at Mount Sinai (Protocols: IACUC-2014-0100 and IACUC-

2014-0061) and comply with the Guide for the Care and use of Laboratory Animals (NIH Publications

No. 8023, 8th edition, 2011). Mice were housed on a 12h light/dark cycle. Mice were fed with regular

chow (PicoLab Rodent Diet 20; LabDiet, St. Louis, MO, USA) and studied in the adult stage. Ehhadh KO

mice were provided by Dr. Janardan K. Reddy (Qi et al. 1999) and backcrossed to a C57BL/6N

background (Violante et al. 2019). The generation and acquisition of the LCAD KO (Acadl KO) mouse

has been described (Houten et al. 2013; Kurtz et al. 1998). The generation of Ehhadh/Acadl double KO

mice (DKO) is described in the Results section. The Acadl allele was genotyped using an SNP-based

genotyping assay (Luther et al 2012) and the Ehhadh allele was genotyped using a PCR assay.

We used three experimental cohorts. For the DKO experiments, the cohort consisted of 32 WT

(16 males, 16 females, average age = 5.4 ± 1.7 months), 33 Ehhadh KO (12 males, 21 females, average

age = 5.6 ± 2.5 months), 12 Acadl KO (2 males, 10 females, average age = 10.3 ± 1.7 months), and 6

DKO mice (3 males, 3 females, average age = 4.3 ± 0.8 months). For the cholesterol synthesis

experiment, the cohort consisted of 11 WT (5 males, 6 females, average age = 3.5 ± 1.0 months) and 12

Ehhadh KO (6 males, 6 females, average age = 3.1 ± 1.3 months) mice. The cohort used for the urinary

DCA data with or without L-AC consisted of 8 WT (1 male, 7 females, average age = 6.1 ± 0.4 months)

and 10 Ehhadh KO (2 males, 8 females, average age = 5.7 ± 0.9 months) mice that were fasted for 8

hours. We also used unpublished plasma acylcarnitine data of a previously reported L-AC cohort that

included WT and Ehhadh KO mice subjected to overnight food withdrawal (Violante et al. 2019).

Overnight food withdrawal was performed by removing the food and placing the mice in a clean

cage at 6 p.m. just before the day/night light cycle switches. Early the next morning, blood glucose was

measured using Bayer Contour blood glucose strips. Spontaneously voided urine was collected by

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scruffing the mice and placing them over a plastic wrap in order to collect the specimen. Mice were

euthanized by exposure to CO2. Immediately afterwards, blood was collected for the preparation of

EDTA plasma. Organs were snap-frozen in liquid nitrogen and stored at -80°C for future analysis. Plasma

was used for acylcarnitine analysis. Urine was used for organic acid analysis to measure DCA content.

2 For cholesterol synthesis, animals were injected with deuterium oxide ( H2O) as 0.9% NaCl at 7

a.m., aiming to reach 4% enrichment of total body water using the following formula: Volume of heavy

water injected (mL) = Body weight (g) * 0.75 (Water content in the body) * 0.04 (% of enrichment). After

2 7 hours, a small amount of blood was drawn from the animals to measure H2O enrichment, and

immediately after, the mice were euthanized by exposure to CO2. The liver was quickly frozen with a

freeze-clamp, wrapped in aluminum foil, temporarily stored in liquid nitrogen, and ultimately transferred

and stored at -80°C.

For the L-AC experiments, animals received an intraperitoneal injection of vehicle (0.9% NaCl)

or L-AC (16 mg/kg) at 6 p.m., before the 12-hr dark cycle, followed by overnight food withdrawal. In a

second experiment, animals received either vehicle or L-AC (16 mg/kg i.p.) at 9 a.m., followed by food

withdrawal for 8 hours. Urine, blood and organs were collected as described above.

Cell culture and incubation with fatty acids

HEK-293 cells were cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and

100 mg/mL streptomycin, in a humidified atmosphere of 5% CO2, at 37°C. The L-AC treatment and

subsequent acylcarnitine profiling after lauric acid loading in HEK-293 cells were performed as

previously described (Chegary et al. 2008; Violante et al. 2013, 2019). For the measurement of DCA

metabolism, HEK-293 cells were seeded in 6-well plates (0.8 x 106 cells per well) and incubated

overnight at 37°C. The following day, the medium was removed and 2 mL of the incubation mixture were

added to each well. Incubation mixtures consisted of DMEM (with 10% FBS and antibiotics)

supplemented with 250 µM hexadecanedioic acid (C16-DCA, added from a 25 mM stock in 100%

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ethanol) or vehicle. After incubation for 72 hours in a humidified CO2 incubator (5% CO2, 95% air) at

37°C, the medium was collected and cells were washed and resuspended in 100 µL of RIPA buffer.

Protein content was measured using the bicinchoninic acid (BCA) assay and human serum albumin as

standard.

EHHADH overexpression

Control and HSD17B4 KO cell lines were transfected with a plasmid containing the human

EHHADH cDNA (MGC premier cDNA clone for EHHADH, pCS6 (BC038948), Transomic) or an empty

vector. Transfection was performed with Lipofectamine 2000 following the supplier´s instructions. After

24-hr incubation, cells were incubated with lauric acid +/- L-AC in one experiment, and C16-DCA in the

other experiment. The medium was analyzed after 72-h incubation as previously described.

Overexpression of EHHADH was confirmed by immunoblot.

Acylcarnitine profiling

HEK-293 cells, liver slices culture medium, and mouse plasma acylcarnitines were measured

after derivatization to butylesters according to a standardized protocol (Violante et al. 2019). Tissue

acylcarnitines were measured in freeze-dried tissue samples (approx. 50 mg of liver) after derivatization

to propylesters essentially as described (Ranea-Robles et al. 2020; van Vlies et al. 2005).

Organic acid analysis

Organic acids from mouse urine, HEK-293 cell culture medium, and mouse liver slices culture

medium were analyzed essentially as previously described with minor modifications (Leandro et al.

2020). Urine specimens were normalized to creatinine (creatinine range between 0.1 and 1 µmol).

Organic acids were oximated prior to acidification and extraction. Quantification of 3-hydroxy DCAs was

performed using a calibration curve of a mix containing 3-OH-adipic, 3-OH-suberic, 3-OH-sebacic and 3-

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OH-dodecanedioic acid. 3-OH-adipic acid was quantified as the sum of the open chain form and the

cyclic lactone form (3-hydroxyadipic acid 3,6-lactone).

Generation of CRISPR-Cas9 KO cell lines

The generation of gene KO HEK-293 cell lines using the CRISPR-Cas9 genome editing

technique was performed as previously described (Violante et al. 2019). For each gene, 2 guides were

designed (Table S1). We selected 2 independent clonal KO cell lines for each targeted gene (Violante et

al. 2019). A list of the newly established cell lines for this study is provided in Table S1.

Immunoblot analysis

Cells and tissues were lysed in RIPA buffer supplemented with protease and phosphatase

inhibitors (Thermo Fisher Scientific), followed by sonication and centrifugation (10 min at 12,000 rpm at

4°C). Protein concentration was determined by the BCA method. Proteins were separated on BoltTM 4-

12% Bis-Tris Plus gels (Invitrogen, Thermo Fisher Scientific), blotted onto a nitrocellulose membrane

(926-31092, LI-COR) and detected using the following primary antibodies: anti-ACOX1 (ab184032,

Abcam), anti-ACAA1 (12319-2-AP, Proteintech), anti-EHHADH (GTX81126, Genetex), anti-HSD17B4

(ab128565, Abcam), anti-HMGCR (AMAb90618, Atlas Antibodies), anti-citrate synthase (GTX628143,

Genetex), and anti-α-tubulin (32-2500, Thermo Fisher). Anti-MVK and anti-PMVK antibodies were a gift

from Dr. Hans Waterham (AMC, Amsterdam) (Hogenboom et al. 2004b,a). Proteins were visualized with

goat anti-mouse and goat anti-rabbit secondary antibodies IRDye 800CW and IRDye 680RD (LI-COR,

926-32 210, 926-68 070, 926-32 211, 926-68 071). Band intensities were quantified using the Fiji

distribution of ImageJ 1.x (Schindelin et al. 2012).

2 Measurement of cholesterol synthesis and de novo lipogenesis by the H2O method

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The contribution of cholesterol synthesis (2H-labeled cholesterol) to the pool of total hepatic

cholesterol and the contribution of de novo lipogenesis (2H-labeled TG-bound palmitate, oleate and

2 stearate) to the pool of total hepatic TG-bound fatty acids in mice labeled with H2O were determined

using gas chromatography-mass spectrometry (GC-MS), as previously described (Bederman et al. 2009;

Brunengraber et al. 2003). These measurements were performed at the Metabolic Phenotyping Mass

Spectrometry Core (MPMS), UTHSC, Memphis, TN.

Metabolic tracing of [U-13C]-C12-DCA in precision-cut mouse liver slices

Mouse livers were isolated from female fed WT and Ehhadh KO adult mice (n = 5) between 7

and 11 a.m. Precision-cut liver slices (diameter 8 mm, thickness 250 µm) were prepared in ice-cold

Krebs-Henseleit buffer saturated with carbogen (95% O2 / 5% CO2), using the Leica VT1200 S vibrating

blade microtome with Vibrocheck as previously described (De Graaf et al. 2010; Zimmermann et al.

2009). After slicing, samples were preincubated 1 hour in WEGG medium, and randomly selected for the

vehicle or [U-13C]-C12-DCA treatment. Slices were then incubated 24 hours in cell culture inserts in 6-

well plates with fresh WEGG medium containing 0.4 mM carnitine and either 250 µM [U-13C]-C12-DCA

or vehicle. [U-13C]-disodium dodecanedioate was prepared by dissolving the acid in ethanol followed by

the addition of 1M NaOH in 80% methanol (2 times the equimolar amount). After evaporation of the

solvent, the DCA tracer was dissolved in the WEGG medium. Liver slices were stored at -80°C and the

medium was stored at -20°C for metabolite analysis.

13C-labeling patterns of C6-DCA, C8-DCA, 3-OH-C8-DCA, C6DC-carnitine, C8DC-carnitine,

citric acid cycle intermediates, amino acids, and the ketone body β-hydroxybutyrate (β-OHB) were

analyzed from the culture medium and liver slice homogenates using stable isotopomer mass

spectrometry analysis, as previously described (Zhang et al. 2015). Concentrations of universally 13C

labeled DCAs and 3-hydroxy DCAs (M+6 in the case of C6-DCAs, and M+8 in the case of C8-DCAs) in

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culture medium were quantified using the same calibration curves used for the quantification of the

correspondent non-labeled metabolites.

The fractional isotopic enrichments (via 13C label incorporation) for citric acid cycle

intermediates and β-OHB were defined as molar percent enrichment (MPE) after background correction

using a matrix method, as previously described (Zhang et al. 2015). Briefly, the MPE (unit: percent) is

calculated as:

Ai MPE mn 100 x n ∑i0 Ai

13 where MPE(M+n) is the nth C-labeled mass isotopomer enrichment of each metabolite. A is the

abundance (arbitrary unit). The variable i varied from zero to n, while n is no more than 6. A(0) is the

abundance for the non-labeled metabolite, and A(i) is the abundance for mass isotopomer labeled with i

13C, regardless of position (Zhang et al. 2015).

RNA-seq, differential gene expression, pathway enrichment

RNA was isolated using QIAzol lysis reagent, followed by purification using the RNeasy kit

(Qiagen). RNA samples were submitted to the Genomics Core Facility at the Icahn Institute and

Department of Genetics and Genomic Sciences, and sequenced essentially as previously described

(Argmann et al. 2017). Briefly, mRNA-focused cDNA libraries were generated using Illumina reagents

(polyA capture), and samples were run on an Illumina HiSeq 2500 sequencer to yield a read depth of

approximately 43 million 100 nucleotide single-end reads per sample. Reads from fastq files were aligned

to the mouse genome mm10 (GRCm38.75) with STAR (release 2.3.0e) (Dobin et al. 2013) and

summarized to gene- and exon- level counts using featureCounts (Liao et al. 2014). Differential gene

expression analysis was conducted with R packages limma (Law et al. 2014; Ritchie et al. 2015) and

DESeq2 (Love et al. 2014) as previously described (Argmann et al. 2017). The cut-off for differential

gene expression was chosen at an adjusted p-value (Benjamini-Hochberg) of < 0.05. Gene Set

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Enrichment Analysis (GSEA) using the Molecular Signatures Database (MSigDB) was performed to

determine whether or not a specific subset of genes were enriched in Ehhadh KO livers (Subramanian et

al. 2005).

Statistical analysis

Data are displayed as the mean ± the standard deviation (SD) or the standard error of the mean

(SEM) as indicated in the figure legends. In most cases, individual values are shown. Differences were

evaluated using unpaired t-test with Welch’s correction, one-way ANOVA with Tukey´s multiple

comparison test, or two-way ANOVA, as indicated in the figure legends. If one or more groups were not

distributed normally, non-parametric Kruskal-Wallis test with Dunn´s multiple comparison posthoc test

was used. Significance is indicated in the figures. GraphPad Prism 8 was used to compute statistical

values.

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Results

Generation and validation of HEK-293 cell lines with peroxisomal enzyme deficiencies

We previously used CRISPR-Cas9 genome editing in HEK-293 cells to demonstrate that

peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and

HSD17B4 (Violante et al. 2019). In our assay, carnitine palmitoyltransferase 2 (CPT2) inhibition using L-

AC in combination with lauric acid (C12) loading is used to unveil the production of peroxisomal

acylcarnitines including C10-carnitine. A defect in peroxisomal biogenesis (PEX13 KO) or peroxisomal

fatty acid transport (ABCD3 KO) abolishes the production of the peroxisomal acylcarnitines (Violante et

al. 2019). In a set of newly generated KO cell lines (Table S1), we now study each step in peroxisomal

FAO in more detail. We demonstrate that ACOX1 is essential for the production of C10-carnitine, and

thus catalyzes the first step of the peroxisomal oxidation of C12-CoA (Fig. 1A). For the fourth and last

step of the peroxisomal oxidation of lauric acid, both ACAA1 and SCPx (encoded by the SCP2 gene)

appear non-essential as the single KO cell lines had no defect in C10-carnitine production (Fig. 1A).

ACAA1/SCP2 DKO cell lines, however, failed to accumulate C10-carnitine showing that these enzymes

are redundant in the peroxisomal oxidation of lauric acid (Fig. 1A).

We also studied in more detail the role of EHHADH and HSD17B4. We previously demonstrated

that HSD17B4, but not EHHADH, is essential in the peroxisomal β-oxidation of lauric acid in HEK-293

cells (Violante et al. 2019). The HSD17B4 KO cell lines, however, produced small amounts of C10-

carnitine (Fig. 1A). Therefore, we hypothesized that despite its low expression in HEK-293 cells,

EHHADH can partially compensate for the loss of HSD17B4 (Violante et al. 2019). To test this

hypothesis, we generated EHHADH/HSD17B4 DKO cell lines. Production of the peroxisomal C10-

carnitine was fully defective in EHHADH/HSD17B4 DKO cell lines (Fig. 1A), mirroring the profile of

PEX13 KO, ABCD3 KO, ACOX1 KO and ACAA1/SCP2 DKO cell lines. This result indicates that,

depending on its expression level, EHHADH may be able to take over the function of HSD17B4 in lauric

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acid oxidation. To further demonstrate this concept, we overexpressed EHHADH in EHHADH/HSD17B4

DKO cell lines. EHHADH overexpression rescued defective peroxisomal C10-carnitine production upon

L-AC treatment (Fig. 1B). Combined, these results indicate that EHHADH and HSD17B4 are

functionally redundant in lauric acid oxidation, and their individual roles may in part be determined by

tissue expression levels.

Hexadecanedioic acid is β-oxidized in the peroxisome of HEK-293 cells

Next, we explored if HEK-293 cells can be used to study the metabolism of DCAs. The

generation of DCAs depends on the expression of members of the cytochrome P450 4A family, which is

restricted to liver and kidney (Hardwick et al. 1987; Mortensen 1992; Uhlén et al. 2015). Accordingly,

expression of CYP4A11 and CYP4A22 is very low in HEK-293 cells (Uhlén et al. 2015). We therefore

supplied HEK-293 cells directly with hexadecanedioic acid (C16-DCA) and dodecanedioic acid (C12-

DCA). For both DCAs we observed production of chain-shortened DCAs in the cell culture media

demonstrating that DCA β-oxidation occurs in HEK-293 cells (Fig. 1C, D and data not shown). We next

loaded all our mutant cell lines with C16-DCA and determined DCA chain shortening through β-

oxidation. DCA chain shortening was defective in PEX13 KO cell lines, but not affected in CPT2 KO cell

lines (Fig. 1C). This observation is consistent with the notion that DCA β-oxidation occurs primarily in

peroxisomes and not in mitochondria.

We then studied the contribution of candidate peroxisomal transporters and enzymes to DCA β-

oxidation. We observed deficient DCA β-oxidation in ABCD3 KO and ACOX1 KO cell lines (Fig. 1D).

Production of chain-shortened DCAs was also severely reduced in HSD17B4 KO cell lines, whereas it

was not affected in EHHADH KO cell lines (Fig. 1D). This is surprising as all currently available

evidence clearly implicates EHHADH in DCA metabolism (Ding et al. 2013; Dirkx et al. 2007;

Ferdinandusse et al. 2004; Houten et al. 2012; Nguyen et al. 2008). As discussed above for the oxidation

of lauric acid (Fig. 1A, B), this finding may be related to the low expression level of EHHADH in HEK-

14 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

293 cells (Violante et al. 2019). Notably, some C14-DCA remained detectable in HSD17B4 KO cell lines

indicating a potential small contribution of EHHADH (Fig. 1D). Indeed, EHHADH/HSD17B4 DKO cell

lines were completely defective in C16-DCA β-oxidation (Fig. 1E). To overcome the limitation of low

EHHADH expression in HEK-293 cells, we overexpressed EHHADH in HSD17B4 KO cells and

evaluated C16-DCA β-oxidation. EHHADH overexpression rescued the defective C16-DCA β-oxidation

in HSD17B4 KO cells (Fig. 1E, Supplementary Fig. S1B). Moreover, EHHADH overexpression slightly

increased C6-DCA production in control HEK-293 cells (Fig. 1E). Combined, these data illustrate that

both EHHADH and HSD17B4 are able to handle DCAs and that their role may be mainly driven by

tissue expression levels.

Production of C14-, C12- and C10-DCA was not affected in ACAA1 KO cell lines, but the levels

of C8- and C6-DCA were clearly decreased compared with control cells (Fig. 1D). This indicates that

ACAA1 may have a substrate preference for medium-chain DCAs and would implicate SCPx in the

metabolism of long-chain DCAs. Unexpectedly, DCA β-oxidation was normal in SCP2 single KO cells

(Fig. 1D). DCA β-oxidation was completely defective in ACAA1/SCP2 DKO cells (Fig. 1D). These

results show that ACAA1 and SCPx have overlapping functions in DCA metabolism, with a more

important role of ACAA1 in the β-oxidation of medium-chain DCAs.

15 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

EHHADH plays an important role in medium-chain DCA metabolism

The role of EHHADH in DCA β-oxidation is well documented (Ding et al. 2013; Dirkx et al.

2007; Ferdinandusse et al. 2004; Houten et al. 2012; Nguyen et al. 2008), but the data on the levels of

DCAs in liver tissue and urine are not consistent. Ding et al reported an accumulation of DCAs (C6- to

C16-DCA) in the liver of Ehhadh KO mice (Ding et al. 2013). In contrast, Houten et al reported

decreased C6DC-carnitine in plasma, liver and urine as well as decreased urinary adipic acid (C6-DCA)

excretion in Ehhadh KO mice (Houten et al. 2012). However, the urine organic acid analysis was

hampered by a lack of sample volume (Houten et al. 2012). To further clarify the consequences of

EHHADH deficiency on urinary excretion of DCAs, we now provide a systematic quantification of

DCAs (C6-, C8-, C10- and C12-DCA as well as their 3-hydroxy intermediates) in the urine of Ehhadh

KO mice. To increase the flux through the ω-oxidation and peroxisomal FAO pathways, we collected the

samples after food withdrawal as well as from mice with pharmacological (L-AC) or genetic inhibition

(Acadl KO) of mitochondrial FAO.

Ehhadh+/- animals were crossed with Acadl+/-animals (both on a pure C57BL/6 background) to

generate double heterozygous animals. A cross of double heterozygous mice did not produce the expected

number of progeny, with less Ehhadh/Acadl double KO (DKO) animals than expected (Table S2A).

Similarly, a cross of Ehhadh-/-Acadl+/- x Ehhadh-/-Acadl+/- mice had a less than expected number of DKO

animals (Table S2B). Gestational loss is part of the phenotype of the Acadl KO mouse model (Berger &

Wood 2004; Kurtz et al. 1998) but has not been observed in the Ehhadh KO mouse model (Qi et al.

1999). Using different breeding strategies, we ultimately obtained 6 DKO animals. These mice appeared

healthy. Upon overnight food withdrawal, DKO animals were hypoglycemic, similarly to Acadl KO mice

(Fig. 2A). Increases in the liver and heart relative weights were more pronounced in the DKO mice (Fig.

2A).

We measured the acylcarnitine profile in the plasma (Table S3A) and liver (Table S3B) of these

mice. A reproducible metabolite marker of Ehhadh KO and DKO mice was the increase in the ratio

16 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

between C8DC- and C6DC-carnitine in plasma (Fig. 2B) and liver (Fig. 2C), which is mostly driven by

decreased C6DC-carnitine levels. The ratio of these two metabolites can be viewed as a proxy for the β-

oxidation of C8-DCA into C6-DCA. These data are consistent with the role of EHHADH in DCA

metabolism and replicates our previous findings (Houten et al. 2012). Acadl KO mice present a

characteristic acylcarnitine profile caused by the defect in mitochondrial FAO with a prominent elevation

of C14:1-carnitine, accompanied by secondary carnitine deficiency (Bakermans et al. 2013; Kurtz et al.

1998; Ranea-Robles et al. 2020). However, no major differences were detected in the plasma or liver

acylcarnitine profile of DKO mice compared with Acadl KO mice (Table S3A, S3B). Similar changes

were observed in the acylcarnitine profile of Ehhadh KO mice after mitochondrial FAO inhibition with L-

AC (Violante et al. 2019). We observed an increase in C8DC-/C6DC-carnitine in the plasma and liver of

L-AC-treated Ehhadh KO mice (Supplementary Fig. S2A, S2B). Overall, the consequences of a

combined EHHADH and LCAD defect appear limited to DCA metabolism, underscoring the importance

of EHHADH and peroxisomes in this metabolic pathway.

Medium-chain dicarboxylic aciduria is a prominent finding in the urine from fasted mice,

especially in mice with a defect in mitochondrial FAO, including the Acadl KO mouse (Cox et al. 2001).

We found elevated adipic acid (C6-DCA) levels in the urine of fasted Ehhadh KO, Acadl KO and DKO

mice. The excretion of adipic acid was highest in Acadl KO and DKO mice (Fig. 2D). Although suberic

acid (C8-DCA) was already increased in fasted Acadl KO mice, this increase was very notable in fasted

DKO mice (Fig. 2D). Consequently, there was a significant increase in the ratio between urinary C8-DCA

and C6-DCA in fasted DKO mice compared with single Ehhadh KO or Acadl KO mice (Fig. 2D). We

also detected elevated levels of sebacic acid (C10-DCA) and dodecanedioic acid (C12-DCA) in the urine

of fasted DKO mice (Supplementary Fig. S2C). The increase in adipic acid in the urine of Ehhadh KO

mice is consistent with the reported elevation of this metabolite in liver extracts (Ding et al. 2013).

Even more remarkable changes were observed for the 3-hydroxy DCAs. 3-Hydroxyadipic acid

(3-OH-C6-DCA) was elevated in the urine of fasted Ehhadh KO and Acadl KO mice, but most

prominently in DKO mice (Fig. 2D). Although already increased in fasted Ehhadh KO mice, 3- 17 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

hydroxysuberic (3-OH-C8-DCA) and 3-hydroxysebacic acid (3-OH-C10-DCA) levels were very high in

DKO mice (Fig. 2D and Supplementary Fig. S2C). 3-Hydroxydodecanedioic acid (3-OH-C12-DCA)

levels were generally low and not reliably detected. These findings were replicated in the urine of Ehhadh

KO animals treated with L-AC (Supplementary Fig. S2D). From our data, we conclude that EHHADH

plays an important role in medium-chain DCA metabolism signified by a prominent 3-

hydroxydicarboxylic aciduria and an increased C8DC- to C6DC ratio.

EHHADH deficiency reduces the β-oxidation of [U-13C]-C12-DCA in mouse liver slices

Ding et al. showed a defect in C12-DCA degradation and C6-DCA formation in liver

homogenates from Ehhadh KO mice (Ding et al. 2013). We further investigated this defect using a

precision-cut liver slice (PCLS) culture system. PCLS are viable ex vivo explants of mouse liver that are

metabolically active and reduce the amount of substrate needed for metabolic tracing studies. PCLS also

represent a more relevant physiological model compared with isolated cultured cells and liver

homogenates (De Graaf et al. 2010; van de Bovenkamp et al. 2005). DCA metabolism was probed with

[U-13C]-C12-DCA, which was added to the culture media of the PCLS from WT and Ehhadh KO mice.

We then measured the amount of C6-DCA (M+6), C8-DCA (M+8), and their M+6 and M+8 carnitine

conjugates, which reflect 3 or 2 completed cycles of DCA β-oxidation, respectively. We observed an

increased ratio of [U-13C]-labeled C8- to C6-DCA in the media from Ehhadh KO liver slices (Fig. 3A).

We also observed elevated levels of 13C-labeled 3-hydroxysuberic acid (3-OH-C8-DCA, M+8) in the

media of Ehhadh KO liver slices compared with WT liver slices (Fig. 3B). The ratio of [U-13C]-labeled

C8DC- to C6DC-carnitine tended to increase in the media of Ehhadh KO liver slices compared with those

obtained from WT mice (Fig. 3C). Combined, these data further illustrate the important role of EHHADH

in the hepatic β-oxidation of medium-chain DCAs.

18 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

To reveal the metabolic destination of the DCA carbons after peroxisomal β-oxidation, we

measured the 13C-enrichment in TCA cycle intermediates, amino acids, and the ketone body β-

hydroxybutyrate (β-OHB) in WT and Ehhadh KO liver slices after incubation with [U-13C]-C12-DCA

(Zhang et al. 2015). We observed a 13C-enrichment in acetyl-CoA and TCA cycle intermediates,

including succinate M+2 and M+4 (Supplementary Fig. S3). We noted a high enrichment in β-OHB M+2

(Fig. 3D) closely resembling the enrichment of acetyl-CoA. The 13C-enrichment in β-OHB (M+2 and

M+4) was decreased in Ehhadh KO liver slices (Fig. 3D). These observations suggest that acetyl-CoA

products derived from peroxisomal DCA β-oxidation can be shuttled to mitochondria for further

metabolism such as oxidation or ketogenesis. The deficiency of EHHADH may decrease peroxisomal

acetyl-CoA production from DCAs.

Perturbation of hepatic cholesterol homeostasis in Ehhadh KO mice

To shed more light on the physiological functions of DCA metabolism, we performed RNA

sequencing analysis of RNA isolated from the livers of WT and Ehhadh KO animals after an overnight

food withdrawal. We only found 17 differentially expressed genes (DEG) by DESeq2 (12 up and 5

down), of which 2 were also detected by limma technology (Fig. 4A, Table S4A and S4B). The low

number of DEG is consistent with the absence of detectable phenotypic defects in the Ehhadh KO mouse

(Qi et al. 1999). Interestingly, within the DEG, we noted two genes involved in cholesterol biosynthesis;

Hmgcr (hydroxymethylglutaryl-CoA reductase) and Lss (lanosterol synthase) (Fig. 4A). Next, we used

Gene Set Enrichment Analysis (GSEA), which revealed a significant upregulation of genes involved in

cholesterol homeostasis (Fig. 4B and Table S4C). No other pathways were significantly enriched (Table

S4C).

Given the small fold change of the upregulation of genes involved in cholesterol homeostasis, we

studied protein expression. We found that HMGCR, which catalyzes the rate-limiting step in cholesterol

biosynthesis, was increased 10.2-fold in Ehhadh KO livers (Fig. 4B). The increase in HMGCR protein

19 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

was much larger than the increase in Hmgcr mRNA, which may be explained by well-documented

posttranscriptional regulatory mechanisms of HMGCR (Goldstein & Brown 1990; Jun et al. 2020).

Mevalonate kinase (MVK, 1.5-fold) and phosphomevalonate kinase (PMVK, 1.9-fold) were also

increased in Ehhadh KO livers (Fig. 4B), but the fold-change was much smaller when compared to

HMGCR. The elevation of HMGCR, MVK and PMVK in Ehhadh KO mice was reproduced in an

independent set of WT and Ehhadh KO mice (Supplementary Fig. S4A). HMGCR protein levels were

increased both in male and female Ehhadh KO mice (Supplementary Fig. S4B), as well as in Ehhadh KO

animals treated with L-AC (Supplementary Fig. S4C). These results show that cholesterol homeostasis is

perturbed in Ehhadh KO mice.

To support the role of peroxisomal metabolism in cholesterol homeostasis, we measured de novo

2 lipogenesis (DNL) and cholesterol biosynthesis in WT and Ehhadh KO mice using the H2O method

(Bederman et al. 2009; Brunengraber et al. 2003). We found that the amount of hepatic cholesterol and

the rate of hepatic cholesterol synthesis were sexually dimorphic, with higher rates and increased levels of

cholesterol in female mice (Fig. 4C, D). Cholesterol synthesis was decreased in female Ehhadh KO and

not changed in male Ehhadh KO mice compared to sex-matched controls (Fig. 4C). We did not find any

changes in DNL in Ehhadh KO mice compared with WT mice (Supplementary Fig. S4D, S4E). Thus, the

apparent increase in the mRNA and protein expression of enzymes involved in cholesterol synthesis does

not translate into increased cholesterol synthesis. In fact, cholesterol synthesis was decreased in female

Ehhadh KO mice. These data further indicate a perturbation of cholesterol homeostasis in Ehhadh KO

mice.

20 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Discussion

In this study, we provide a detailed characterization of the contribution of individual peroxisomal

transporters and enzymes to the β-oxidation of DCAs in a human HEK-293 cell line model. We also

provide further evidence supporting the specific role of EHHADH in the hepatic metabolism of DCAs,

and its functional relevance as a modulator of hepatic cholesterol metabolism.

Our study provides more definite genetic proof of the essential role of peroxisomes and the

peroxisomal enzyme EHHADH in DCA β-oxidation, supporting previous research that also pointed to

that direction (Ding et al. 2013; Dirkx et al. 2007; Houten et al. 2012; Nguyen et al. 2008; Poosch &

Yamazaki 1989; Suzuki et al. 1989). Based on our experiment in HEK-293 cells, we propose that the

peroxisomal pathway of DCA β-oxidation is formed by the transporter ABCD3, the acyl-CoA oxidase

ACOX1, the bifunctional enzyme HSD17B4 (with a small contribution of EHHADH due to low

expression), and the thiolases ACAA1 and SCPx. Overexpression of EHHADH can functionally

compensate for HSD17B4 deficiency, indicating that both EHHADH and HSD17B4 are able to handle

DCAs and that their functional role in this process may be mainly driven by tissue expression levels. Our

results also provide evidence for a partially overlapping function of ACAA1 and SCPx in DCA β-

oxidation, with ACAA1 being more important in the β-oxidation of medium-chain DCAs. We speculate

that additional players involved in DCA β-oxidation are a dicarboxylyl-CoA synthetase needed for the

activation of DCAs into DCA-CoAs (ACSL1, acyl-CoA synthetase long chain family member 1) and

subsequent uptake by peroxisomes as well as CROT (peroxisomal carnitine O-octanoyltransferase) and

ACOT4 (Houten et al. 2012, 2020; Hunt et al. 2006; Vamecq et al. 1985; van Roermund et al. 2020;

Westin et al. 2005). The role of these enzymes in DCA metabolism should be addressed in future studies.

Finally, even though our study in HEK-293 cells did not find any role for mitochondrial FAO in DCA β-

oxidation, we cannot exclude that under pathophysiological conditions such as an impairment of

21 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

peroxisomal FAO, DCAs are metabolized in the mitochondria. This hypothesis deserves further

investigation using models of peroxisomal dysfunction.

Remarkably, no human EHHADH defect has been described to date and, therefore, the Ehhadh

KO mouse remains the best tool to study EHHADH’s role in mammalian physiology. We provide

additional evidence for the role of EHHADH in DCA β-oxidation by the identification of medium-chain

3-hydroxydicarboxylic aciduria and an increased ratio of C8-DCA to C6-DCA and C8DC- to C6DC-

carnitine in Ehhadh KO mice. We speculate that a similar metabolite pattern could indicate a human

deficiency in a case with potentially pathogenic biallelic EHHADH variants. However, 3-

hydroxydicarboxylic aciduria by itself is not a specific marker as it frequently accompanies dicarboxylic

aciduria, for example, during fasting (Tserng & Jin 1991b; Tserng et al. 1989). In addition, mitochondrial

long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, as well as other conditions, cause 3-

hydroxydicarboxylic aciduria (Bennett & Sherwood 1993; Bennett et al. 1994; Bergoffen et al. 1993;

Mayatepek et al. 1995; Tserng et al. 1991). Ehhadh KO mice have no obvious hepatic disease phenotype

under normal conditions, but feeding these animals with a diet rich in medium-chain fatty acids led to

liver inflammation, fibrosis, and death (Ding et al. 2013). We speculate that a potential clinical

presentation of human EHHADH deficiency may be hepatic toxicity of medium-chain fat rich foods (e.g.

coconut oil) or supplements such as medium-chain triglyceride oil.

Our results also indicate that DCA β-oxidation is not completely defective in Ehhadh KO mice

but appears to have a limited capacity for medium-chain DCAs. There are several lines of evidence to

support this. First, our in vitro experiments have clearly demonstrated that EHHADH and HSD17B4

display functional redundancy in the metabolism in DCAs, and their contribution to this process appears

to depend on their expression level. This further highlights the role of EHHADH in DCA β-oxidation,

given its high expression levels in liver and kidney. Second, and somewhat counterintuitively, Ehhadh

KO mice are able to generate medium-chain DCAs, including adipic acid, which is considered an end

product of peroxisomal DCA β-oxidation. However, the fact that the highest levels of intermediates are

22 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

observed for suberic acid and 3-hydroxysuberic acid, which are both not typically viewed as final

products of DCA metabolism, suggests that the pathway is saturated. Indeed, the ratio of C8DC over

C6DC is consistently higher in Ehhadh KO animals suggesting that the main bottleneck is at the level of

C8DC β-oxidation. Lastly, stable isotope tracing in liver slices using 13C-C12-DCA as a precursor

reproduced the increased ratio of C8-DCA over C6-DCA for the 13C labeled products in the Ehhadh KO

media compared to WT media. Ultimately our observations are consistent with a model where all DCA β-

oxidation intermediates compete for a common set of enzymes. Such competition becomes problematic at

saturating conditions (van Eunen et al. 2013), which may be because EHHADH is deficient and

HSD17B4 is the only available enzyme and/or because DCA metabolism is induced due to fasting or a

defect in mitochondrial FAO.

Remarkably, liver and plasma C6DC-carnitine levels are generally decreased in Ehhadh KO

mice, whereas urine and liver adipic acid levels are increased. This suggests that adipoyl-CoA metabolism

differs between Ehhadh KO and WT mice. Specifically, conversion to C6DC-carnitine by CROT is

decreased, whereas thiolytic cleavage is increased in Ehhadh KO mice. Given that Ehhadh KO mice do

not display secondary carnitine deficiency, we speculate that increased thiolytic cleavage is the likely

culprit. Indeed, it is known that ACOT8, which displays activity with medium-chain DCAs, is inhibited

by free CoA (Hunt et al. 2002; Westin et al. 2005). We suggest that an accumulation of DCA β-oxidation

intermediates in Ehhadh KO mice due to saturation of the β-oxidation pathway may lead to reduced

availability of peroxisomal free CoA (i.e. CoA trapping), which would stimulate ACOT8 activity and as

such generate higher levels of free DCA intermediates.

The finding that 3-hydroxyadipic acid accumulates in fasted Ehhadh KO, Acadl KO and

Acadl/Ehhadh double KO mice is notable because it indicates that C6-DCA can undergo another round of

peroxisomal β-oxidation, which would involve EHHADH and yield the anaplerotic substrate succinate.

The low but detectable labeling of TCA cycle intermediates after incubating WT and Ehhadh KO liver

slices with [U-13C]-C12-DCA further supports this possibility. The capacity of peroxisomes to generate

23 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

succinate is also supported by the work on ACOT4, a peroxisomal acyl-CoA thioesterase that displays

optimal activity with succinyl-CoA (Hunt et al. 2006; Westin et al. 2005). Even though some studies

using 13C-labeled DCAs were not able to detect metabolism beyond adipic acid (Cerdan et al. 1988), other

studies clearly established anaplerosis from C12-DCA (Jin & Tserng 1991; Jin et al. 2015; Tserng & Jin

1991a). The anaplerotic potential of peroxisomal DCA β-oxidation products in different physiological

situations remains to be established. We provide evidence in this study of the feasibility of mouse liver

slice culture to perform isotope tracer studies to study metabolic reactions, which could be used to

investigate this aspect of peroxisomal metabolism.

Finally, our study shows that EHHADH deficiency perturbs cholesterol biosynthesis. We

observed an induction of cholesterol biosynthesis enzymes at the mRNA and protein level, including a

pronounced increase of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. An increase in the

expression of cholesterol biosynthetic genes is usually mediated by decreased levels of cholesterol and

non-sterol isoprenoids leading to the activation of pathways, such as the proteolytic cleavage of

membrane-bound sterol regulatory element binding protein 2 (SREBP2), which aim to restore cholesterol

levels (Brown & Goldstein 1997). Despite this induction of cholesterol biosynthesis genes, cholesterol

2 biosynthesis, as measured by the H2O method, was reduced in female and not changed in male Ehhadh

KO mice. Combined, these data indicate that cholesterol biosynthesis is decreased due to EHHADH

deficiency leading to a compensatory increase in cholesterol biosynthetic capacity via the SREBP2

pathway.

We propose that peroxisomal β-oxidation is an important source of cytosolic acetyl-CoA that can

be used for cholesterol and fatty acid biosynthesis. Thus, a defect in peroxisomal β-oxidation limits the

availability of this biosynthetic precursor. Peroxisomal acetyl-CoA destined for use in the cytosol is

presumably released in the form of acetate (Kasumov et al. 2005; Leighton et al. 1989), which is

supported by the presence of peroxisomal thioesterases with an affinity for short chain acyl-CoA

molecules such as ACOT8 and ACOT12 (Hunt et al. 2014). Previous studies have shown that

24 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

administration of DCAs leads to cytosolic acetyl-CoA production (Jin et al. 2015; Leighton et al. 1989),

which can be used for the biosynthesis of cholesterol (Dietschy & McGarry 1974; Kondrup & Lazarow

1985). We assessed the fate of acetyl-CoA produced from [U-13C]-C12-DCA in mouse liver slices from

WT and Ehhadh KO mice. Although the acetyl-CoA pool was enriched, we were not able to detect 13C-

enrichment in cholesterol perhaps due to a low overall cholesterol synthesis rate in the mouse liver slices.

We did, however, obtain evidence that peroxisomal DCA β-oxidation provides acetyl-CoA units for other

biochemical pathways such as ketogenesis. The reduced 13C-enrichment observed in the ketone body β-

OHB in Ehhadh KO mouse liver slices compared to WT slices after adding the 13C-C12-DCA tracer

supports the important role of EHHADH in DCA β-oxidation. Even though our results demonstrate that

peroxisomal DCA β-oxidation yields acetyl-CoA that can be used for various metabolic processes, further

experiments in relevant models of peroxisomal FAO deficiencies are warranted to assess this contribution

under different physiologic conditions.

The role of peroxisomes in cholesterol metabolism remains a debated issue. In vitro and in vivo

studies done in different models of peroxisomal deficiency have reported disturbances of cholesterol

homeostasis and activation of the SREBP2 pathway (Charles et al. 2020; Fidaleo et al. 2011; Kovacs et

al. 2004, 2009; Martens et al. 2008; Nicolas-Francès et al. 2014), in line with our findings in the liver of

Ehhadh KO mice. Our observations are consistent with previous reports of reduced cholesterol synthesis

rates in skin fibroblasts from patients with peroxisomal disorders (Hodge et al. 1991). In contrast, other

reports have shown increased rates of cholesterol synthesis in Pex2 KO and Hsd17b4 KO mice (Kovacs et

al. 2004; Martens et al. 2008). In these studies, cholesterol synthesis was increased when measured using

labeled acetate. This increase, however, could be artifactual if the size of the acetate pool differs between

WT mice and mice with a peroxisomal disorder. A decreased acetate pool in mice with peroxisomal

disorders would lead to a higher enrichment with the same dose of labeled acetate, and a perceived

2 increase in cholesterol synthesis. The H2O method that we employed does not have this potential

drawback and directly implicates peroxisomal β-oxidation in the regulation of cholesterol homeostasis.

25 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In summary, our results show that EHHADH has an important role in hepatic DCA metabolism

and highlight the role of peroxisomal FAO in the regulation of cholesterol biosynthesis. Further studies

are necessary to understand the physiological significance of these findings.

Acknowledgements

We thank Dr. Hans Waterham for providing the MVK and PMVK antibodies, Jacob Hagen for

the RNAseq analysis, the Flow Cytometry CoRE at the Icahn School of Medicine at Mount Sinai for their

technical assistance with cell sorting, and Purvika Patel and Dr. Brandon Stauffer (Sema4) for their kind

and excellent technical assistance with the clinical biochemical analyses.

Research reported in this publication was supported by the National Institute of Diabetes and

Digestive and Kidney Diseases of the National Institutes of Health under Award Number R01DK113172.

The content is solely the responsibility of the authors and does not necessarily represent the official views

of the National Institutes of Health.

26 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

References

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32 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure legends

Fig. 1. Peroxisomal β-oxidation of lauric acid and hexadecanedioic acid in HEK-293 cells.

A) Production of peroxisomal C10-carnitine (nmol/mg protein/72h) in the extracellular media of KO cell

lines after loading with C12:0 in the presence of increasing concentrations of the CPT2 inhibitor L-AC.

The cell lines are placed in 3 groups; those with no apparent defect (top), those with deficient but

detectable β-oxidation (only HSD17B4 KO), and finally, those with no detectable peroxisomal β-

oxidation (bottom). Note that the currently reported values are 25 times larger than those reported by

Violante et al (Violante et al. 2019). The current values reflect the total production in 500µL media and

not the 20µl specimen used for the MS analysis. B) Rescue of the production of peroxisomal C10-

carnitine (nmol/mg protein/72h) in EHHADH/HSD17B4 double KO (DKO) cell lines by EHHADH

overexpression. C) Production of chain-shortened DCAs (C14 to C6, nmol/mg protein/72h) by control,

CPT2 KO and PEX13 KO HEK-293 cell lines after 72-hr incubation with hexadecanedioic acid (C16-

DCA). D) Production of chain-shortened DCAs (C14 to C6, nmol/mg protein/72h) by control, ABCD3

KO, ACOX1 KO, EHHADH KO, HSD17B4 KO, ACAA1 KO, SCP2 KO, and ACAA1/SCP2 DKO HEK-

293 cell lines after 72-hr incubation with C16-DCA. Note that lines for ABCD3 KO, ACOX1 KO, and

ACAA1/SCP2 DKO almost completely overlap. E) Production of chain-shortened DCAs (C14 to C6,

nmol/mg protein/72h) by control, HSD17B4 KO and HSD17B4/EHHADH DKO HEK-293 cell lines after

72-hr incubation with C16-DCA. Control and HSD17B4 KO cell lines were analyzed with or without

overexpression of EHHADH. Data are displayed as the mean ± SEM (A), individually + the mean (B), or

as the mean ± S.D (C-E) of 2-4 independent experiments. All experiments were carried out with 2 clonal

cell lines.

Fig. 2. EHHADH plays an important role in medium-chain DCA metabolism.

A) Blood glucose levels (mM), and relative liver-to-BW (body weight) and heart-to-BW percentages in

WT (n=6-11), Ehhadh KO (n=12-17), Acadl KO (n=10) and Ehhadh/Acadl double KO mice (DKO, n=6).

B) Plasma C6DC- and C8DC-carnitine (µM), and the C8DC/C6DC-carnitine ratio in WT (n=12), Ehhadh

33 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

KO (n=17), Acadl KO (n=10) and DKO mice (n=6). C) Hepatic C6DC- and C8DC-carnitine (pmol/mg

dry weight tissue), and the C8DC/C6DC-carnitine ratio in WT, Ehhadh KO, Acadl KO, and DKO mice,

n=4 per genotype. (D) Urinary C6-DCA, C8-DCA, their 3-OH forms (mmol/mol creatinine), and the C8-

DCA/C6-DCA ratio in WT (fed=20, fasted n=12), Ehhadh KO (fed=21, fasted n=12), Acadl KO (fed=6,

fasted n=6) and DKO mice (fed=2, fasted n=3). Data are represented individually with the mean ± S.D.

Statistical significance was tested using one-way ANOVA (A-C), two-way ANOVA with “Genotype”

and “Fed/Fasted” as the two factors (D), or non-parametric Kruskal-Wallis test (C6DC- and C8DC-

carnitine in B). After one-way ANOVA, Tukey’s multiple comparison test was performed *P < 0.05; **P

< 0.01; ***P < 0.001. After non-parametric Kruskal-Wallis test, Dunn’s multiple comparison test was

performed. # P < 0.05

Fig. 3. Metabolic tracing of [U-13C]-C12-DCA in precision-cut mouse liver slices

(A-C) Measured [U-13C]-labeled C6-DCA, C8-DCA, and 3-OH-C8-DCA (µmol/L) in media after 24-hr

incubation of WT and Ehhadh KO liver slices with [U-13C]-C12-DCA, and calculation of the

corresponding C8DCA- to C6-DCA (A) and C8-DC- to C6-DC-carnitine (B) ratios. D) MPE (molar

percent enrichment) of individual 13C-labeled mass isotopomers of β-OHB in liver slices after 24-hr

incubation with [U-13C]-C12-DCA. Data are represented individually with the mean ± S.D. Statistical

significance was tested using unpaired t-test with Welch’s correction. *P < 0.05; **P < 0.01

Fig. 4. Perturbation of hepatic cholesterol homeostasis in Ehhadh KO mice. A) Venn diagram comparing the number of DEG detected by both DESeq2 and limma technologies. All

gene names are provided, and those belonging to the cholesterol biosynthesis pathway are highlighted. B)

Enrichment plot for the HALLMARK CHOLESTEROL HOMEOSTASIS gene set in the molecular

signature database (M5892). The graph shows the profile of the running Enrichment Score (ES) and the

positions of gene set members on the fold-change rank-ordered list. Overall the NES was 2.338538 with

an FDR q-value of 0. C) Immunoblots for HMGCR, MVK, PMVK and the loading control citrate

synthase (CS) in WT and Ehhadh KO mice (n=4 per genotype), and the corresponding quantification. D)

34 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Cholesterol synthesis was calculated from the incorporation of deuterium isotopes (2H) into cholesterol in

the liver. E) Hepatic cholesterol content (mg/g tissue) was measured by mass spectrometry. Data are

represented individually with the mean ± SD. Statistical significance was tested using unpaired t-test with

Welch’s correction (C) or two-way ANOVA with “Genotype” and “Sex” as the two factors followed by

Tukey's multiple comparisons test (D, E). *P < 0.05; **P < 0.01; ***P < 0.001.

35 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.02.433634; this version posted March 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.